Human cell lines have been used in a variety of laboratory applications. For example, immortalized human liver cells have been used in metabolic studies with different chemical classes of carcinogens. Bronchial epithelial cells have been used in studies of the control of squamous differentiation, and identification of chemical and biological agents which induce squamous differentiation. Both of these human cell types have been used in screening chemicals suitable for the treatment of cancer and related diseases, by growing them in vitro in medium containing the chemical to be tested and then, after a suitable period of exposure, determining whether and to what extent genotoxicity, DNA adduct formation, mutagenicity, cell transformation and/or cytotoxicity has occurred following exposure to a carcinogen, e.g., by trypan blue exclusion assay or related assays. These cell lines have also been used for identifying agents that induce programmed cell death or apoptosis, which may have an important impact on prevention of malignant transformation. Programmed cell death is assayed by DNA fragmentation or cell-surface antigen analysis.
Human cell lines have also been used to study DNA mutagenesis. Substances known or suspected to be mutagens, or precursors of mutagens, may be added to the growth medium of the cells and then mutations may be assayed, e.g., by detection of the appearance of drug resistant mutant cell colonies. Similarly, cell-mediated DNA mutagenesis has been investigated by co-cultivating the cells with cell types known or suspected to be capable of secreting mutagenic compounds. Human cell lines have also been used in studies of chromosome damaging agents, studies of malignant transformation, screening for potential chemotherapeutic agents, studies of cellular biochemistry, studies of cellular responses to growth factors and production of growth factors, studies of intracellular communication, characterization of cell surface antigens, hybrid studies for identification of tumor suppressor activity, and identification of novel genes.
In all of the aforementioned studies, the human cell lines used are capable of expressing a cytochrome P450. Cytochromes P450 are a large family of hemoprotein enzymes capable of metabolizing xenobiotics such as drugs, carcinogens and environmental pollutants as well as endobiotics such as steroids, fatty acids and prostaglandins. Some members of the cytochrome P450 family are inducible in both animals and cultured cells, while other constitutive forms are non-inducible. This group of enzymes has both harmful activities, such as the metabolic conversion of xenobiotics to toxic, mutagenic and carcinogenic forms, and beneficial activities, such as the detoxification of xenobiotics.
In the pharmaceutical industry, screening drug candidate compounds for toxicity is a critical step in the drug development process. It is highly desirable to identify, as early as possible, compounds that have an increased likelihood of toxicity. For example, liver toxicity, such as acute liver failure, is currently the leading cause of drug removal from the market.
Chemical injury to the liver is a multi-faceted phenomenon involving factors such as the nature of the toxic agent, the mechanisms of injury, the nature of the exposure and the susceptibility of the host. A variety of agents can lead to hepatic damage, but in general compounds that are able to injure the liver of most recipients in a variety of species are called “true” or “intrinsic” liver toxicants. Agents that depend on unusual susceptibility of the host to unmask their damaging potential are called “idiosyncratic” hepatotoxins. This increased sensitivity of the host's liver to the damaging effects of the chemical can be linked to two broad categories of mechanisms. The first category is accompanied by clinical and physiological symptoms suggesting the involvement of an immune response and is usually designated as “drug hypersensitivity.” The second category consists of liver damage appearing in the absence of concomitant immune reaction, and has been called “metabolic idiosyncrasy” (Zimmerman, H., Hepatoxicity, 2nd ed., Lippincott, Williams & Wilkins, Philadelphia, Pa. (1999)). The hypothesis underlying the “metabolic idiosyncrasy” theory is that some products of drug metabolism are responsible for the damage done to the liver cells, but that those metabolites are not produced in sufficient quantities in the majority of the population to result in overt hepatic injury. In some patients, however, the metabolic pathway of the drug favors the production of a toxic species, resulting in liver injury.
There are many obstacles to predicting metabolite-related liver toxicity of pharmaceuticals. Prediction of liver toxicity in clinical trials is poor due to a number of factors, including high polymorphism in the population, differences in metabolism between animal models and humans, and differences in cytochrome P450 expression due to gender and age. In addition, in vivo studies are expensive and time-consuming.
One of the major obstacles in the drug development process is predicting which drug candidates will exhibit idiosyncratic toxicity. A major challenge in predicting metabolic idiosyncrasy is not to find models that are representative of the majority of the population, but rather to find models that are representative of those few subjects for which the metabolism is oriented toward toxicity. As compared to intrinsic toxicity, idiosyncratic toxicity is far more difficult to predict, due to the nature of the toxicity. Intrinsic toxicity is: (1) typically a property of the drug; (2) a constant occurrence; (3) dose-dependent; (4) characterized by a clear onset of toxicity; and (5) predicted by animal models. Idiosyncratic toxicity, on the other hand, is: (1) typically a result of interaction between the drug and patient specific factors; (2) a relatively rare occurrence (<1/10,000); (3) usually independent of dose; (4) characterized by a delayed onset; and (5) difficult (if not impossible) to predict with animal models.
Thus, there is a need for rapid, affordable, accurate methods of screening compounds for toxicity. Specifically, there is a need for methods to predict idiosyncratic toxicity of a drug candidate. In particular, there is a need for methods to identify idiosyncratic toxicity early in the drug development process. There is also a need for methods of screening compounds for toxicity which reflect human metabolism and are representative of the diverse human population.